Targeting of drugs or genetic material to defined cells, tissues or organs increases the specificity and effectiveness of drug therapy and reduces the incidence of potentially harmful side effects. Intracellular delivery and proper intracellular processing are required for specific and effective therapeutic applications of certain classes of drugs including, but not limited to, immunotoxins, antioxidants, NO-donors, antibiotics, antisense oligonucleotides, nucleic acids and intracellular hormones. Further, intracellular delivery of gene therapy products is crucial to successful treatment.
In the case of antioxidants, immunotoxins, antisense agents, hormones, gene therapy agents and other therapeutic compounds, referred to herein as “effectors”, only limited spontaneous cellular internalization typically occurs. Accordingly, strategies to facilitate or enhance internalization have been developed and include chemical modification with polyethylene glycol (Abuchowski et al. J. Biol. Chem. 1977 252(11):3852–3586; Abuchowski et al. J. Biol. Chem. 1992 252(11):3578–3581; Beckman et al. J. Biol. Chem. 1988 263:6884–6892), encapsulation in liposomes (Freeman et al. J. Biol. Chem. 1983 258:12534–12542; Briscoe et al. Am. J. Physiol. 1995 12(3):L374–L380), and conjugation with ligands of internalizable receptors (Wagner et al. Adv. Drug. Del. Rev. 1994 14:113–135; Chen et al. FEBS Lett. 1994 338:167–169).
Although these strategies may facilitate internalization, their applicability is restricted. For example, none of these methods provides targeting of an effector to a specific cell, tissue, or organ, restricting the specificity and safety of the therapeutic agent. Further, these methods utilize cellular mechanisms of internalization leading to accumulation of an effector in the lysosomes and ultimately resulting in degradation and inactivation of the effector compound.
Antibodies recognizing cell-specific surface determinants are useful for targeting compounds to defined cells, tissues, or organs. Chemical conjugation of a cell-specific antibody with an effector has been investigated as a means to achieve specific targeting (Poznansky M. and Juliano, R. Pharmacol. Rev. 1984 4:278–345). Antibodies capable of effective internalization can provide intracellular delivery of a drug (Raso, V. Anal. Biochem. 1994 222:297–304; Chen et al. FEBS Lett. 1994 338:167–169). For example, studies have been performed targeting drugs conjugated with internalizable antibodies against receptors for transferrin, growth factor and folate (Wagner et al. Adv. Drug. Del. Rev. 1994 14:113–135; Chen et al. FEBS Lett. 1994 338:167–169). Internalizable antibodies, however, underwent massive intracellular degradation in lysosomes (Brisson et al. Throm. Haremost. 1992 68:737–743; Raso, V. Anal. Biochem. 1994 222:297–304; Reilly et al. Clin. Pharmacokinet. 1995 28:126–142; Muzykantov et al. Circulation 1997 8:43–44). Accumulation of these antibodies or antibody-conjugated effectors in lysosomes and subsequent lysosomal degradation restrict the applicability of the internalizable antibody as a carrier for intracellular delivery of drugs.
Other potentially useful antibodies that recognize specific antigens abundant on the surface of target cells are “poorly internalizable” (Matzku et al. Int. J. Cancer 1988 2:11–14; Reilly et al. Clin. Pharmacokinet. 1995 28:126–142). The lack of internalization diminishes intracellular delivery and accumulation in target organs or tissues (Matzku et al. Int. J. Cancer 1988 2:11–14; Reilly et al. Clin. Pharmacokinet. 1995 28:126–142). Therefore, these “poorly internalizable” antibodies are not useful for intracellular targeting.
Among potential target cells, pulmonary vascular endothelium represents an important target for intracellular delivery of drugs, genes, enzymes, NO-donors and other effectors (Erzurum et al. Nucl. Acid Res. 1993 21:1607–1612; von der Leyen et al. Proc. Natl Acad. Sci. USA 1995 92:1137–1141; Gibbons, G. and Dzau, V. Science 1996 272:689–693; Rodman et al. Am. J. Respir. Mol. Cell. Biol. 1997 16:640–649). Several monoclonal antibodies have been studied as potential carriers for intracellular delivery of drugs to endothelial cells. For example, internalizable antibodies against thrombomodulin (Kennel et al. Nucl. Med. Biol. 1990 17:193–200; Maruyama et al. Proc. Natl. Acad. Sci. USA 1990 87:5744–5748) and E-selectin (Kuijpers et al. J. Immunol. 1994 152:5060–69; Spragg et al. Proc. Natl Acad. Sci. USA 1997 94:8795–8800) have both been conjugated to drugs for targeting to endothelial cells. However, these internalizable antibodies underwent massive intracellular degradation in lysosomes.
Another example of an antibody carrier that has been tested as a means of internalizing effectors is antibody to angiotensin-converting enzyme (anti-ACE; Muzykantov, V. et al. Am. Rev. Res. Dis. 1989 136:1464–1473). The methods utilized were based on the conjugation of an effector with anti-ACE, an antibody that recognizes pulmonary endothelial surface antigen (Danilov et al. Lab. Invest. 1991 64:118–124). The anti-ACE carrier provided intracellular targeting (50–60% internalization) and underwent moderate destruction in the lysosomes (15–20% degradation) (Muzykantov et al. Proc. Natl Acad. Sci. USA 1996 93:5213–5218). However, the total amount of anti-ACE binding sites in pulmonary endothelium was limited to 2×105 per cell (Muzykantov et al. Am. J. Physiol. 1996 270:L704–713). The limited number of binding sites, as well as significant intracellular degradation, limit the utility of this antibody system for intracellular targeting. In addition, anti-ACE accumulation in the lung causes suppression of ACE activity in the tissue (Danilov et al. Intern. Immunol. 1994 6:1153–1160). In pathological conditions associated with acute hypotonia, inhibition of ACE activity may lead to dangerous side effects, such as vascular collapse.
Since streptavidin-biotin cross-linker was utilized for conjugation of drugs to anti-ACE, the effects of biotinylation and conjugation with streptavidin on anti-ACE targeting, binding and internalization by endothelium was examined (Muzykantov et al. Anal. Biochem. 1995 226:279–287). These studies showed no significant effect of biotinylation and subsequent conjugation with streptavidin on these parameters.
Accordingly, there is a need for antibody systems which provide intracellular targeting of selected cells with a large amount of effector while escaping the lysosomal degradation pathway.
In the present invention, a method is provided for facilitating intracellular delivery to endothelium of a carrier antibody and antibody-conjugated effectors to pulmonary endothelial cells. Further, this methods has been successfully used in other cell types with several antibodies thus demonstrating that applicability of this strategy is not limited to endothelial cells or specific antibodies. The method of the present invention overcomes problems of poor internalization and intracellular degradation in lysosomes, while allowing use of antibodies with higher numbers of binding sites per cell.